Natural killer (NK) cells are a group of cells having a cytotoxic activity against various tumor cells, virus infected cells and cells having different major histocompatibility antigens, and their activation and inhibition are regulated primarily by receptors that recognize self and nonself.
NK cells are considered to have important functions as one type of lymphocytes involved in natural immunity. Against viral infection, in particular, they play a very important role in immune responses in early infection until acquired immunity is established by T lymphocytes and B lymphocytes.
That is, even if T lymphocytes and B lymphocytes are normal, immunodeficient patients and mice that are deficient in the natural killer function are very easily infected by specific viruses. In recent years, it has been revealed that receptors of NK cells recognize products of specific viruses.
Thus, attempts have been made to effectively utilize various functions of NK cells involved in tumor immunity for tumor treatment and removal of virus infected cells that are supposed to become a source of tumor.
As a method for obtaining NK cells from laboratory animals, a method of collecting cells from the spleen, blood, bone marrow and so forth and purifying them is known. However, since all of these tissues contain NK cells only in low proportions, technical skills and suitable equipment are required to secure NK cells necessary for experiments.
As for methods for producing or proliferating NK cells, it is well known that if peripheral blood mononuclear cells are cultured in a culture broth to which a large amount of interleukin-2 is added, lymphokine-activated killer lymphocytes (LAK cells) are proliferated within about 1 week in the case of human, and a large amount of NK cells are contained in this culture. Recently, a method has been developed which comprises irradiating cells of the human B cell strain 721.221, in which the major histocompatibility class I (MHC-I) is hardly expressed on the cell surfaces, with a radioactive ray so that the cells should lose division potential, culturing the cells with monocular cells in peripheral blood as mixed culture for 5 to 6 days, purifying NK cells from the culture and further continuing culture to obtain a large amount of NK cells (e.g., Non-patent document 1).
Further, a method for proliferating human NK cells comprising the step of mixed culture of peripheral blood mononuclear cells and cells of human fibroblast Wilms' tumor line HFWT (Patent document 1), a method for enhancing an activity of animal natural killer lymphocytes utilizing conjugated linoleic acids as an active ingredient (Patent document 2) and so forth have been disclosed. However, the technique disclosed in Patent document 1 is a method for proliferating human NK cells by mixed culture of peripheral blood mononuclear cells and cells of human fibroblast Wilms' tumor line HFWT, and any method for collecting NK cells proliferated in vivo and screening for a factor that enhances the natural killer activity are not disclosed in this document. Further, in Patent document 2, changes in killer lymphocytes enhanced by conjugated dienoic linoleic acids in the spleen was followed, and any method for collecting NK cells proliferated in vivo and screening for a factor that enhances the natural killer activity are not disclosed in this document.
At present, the most common method for measuring an activity of NK cells (natural killer activity, NK activity) is a method of observing a cytotoxic activity against human K562 cells or mouse lymphoma cell line Yac-1 by co-culturing a group of cells including Yac-1 cells radiolabeled with 51Cr or the like and measuring radioactivity released in the culture supernatant. Further, a method for measuring the NK activity in living bodies is a method of transplanting tumor cells radiolabeled in the same manner as mentioned above into a living body or infecting an animal with a virus or the like and observing the NK activity, and these methods require a special facility and equipment.
Lactoferrin is known as a lactoprotein having various actions such as an antibacterial action, immunity activating action and antitumor action. Lactoferrin is a milk-derived glycoprotein that is highly safe and can be continuously taken for a long period of time. Since lactoferrin itself has almost no taste or odor, it is a highly versatile protein as an additive for various food, drugs and feeds.
Patent document 3 (International Patent Publication in Japanese (Kohyo) No. 2002-515893) proposes a method for stimulating NK cells in a patient which comprises the step of administering a composition containing a modified human lactoferrin to the patient. However, Patent document 3 does not describe the effect of the modified human lactoferrin for actually stimulating NK cells, and its basis has been unclear.
The Toll-like receptors (TLR) recognize various components of bacteria and are believed to play an important role not only in recognition of bacteria in natural immunity but also in activation of acquired immunity, and also considered to have functions of recognizing various pathogenic components and inducing unique responses. Currently, the Toll-like receptor family in mammals consists of 10 family members, and pathogen-constituting components (ligands) recognized by each Toll-like receptor have been identified. These ligands, the pathogen-constituting components, include lipids, sugars, proteins, nucleic acids and so forth (Non-patent documents 2 and 3).    [Patent document 1] Japanese Patent Laid-open (Kokai) No. 2001-149069    [Patent document 2] International Patent Publication in Japanese (Kohyo) No. 2001-503430    [Patent document 3] International Patent Publication in Japanese (Kohyo) No. 2002-515893    [Non-patent document 1] Proc. Natl. Acad. Sci., USA, Vol. 94, 1997, pp. 13140-13145    [Non-patent document 2] Molecular Medicine, Vol. 39, 2002, pp. 238-246    [Non-patent document 3] Molecular Medicine, Vol. 40, 2003, pp. 186-193